Half a century ago, Linus Pauling first showed that sickle cell anemia is a molecular disease (Pauling, 1949; for full citations see list in Section 8, infra). It was later demonstrated that the disease originated from a missense mutation within the β-globin gene, leading to the substitution of valine for glutamic acid on the outer surface of the globin molecule. This amino acid substitution renders the sickle cell hemoglobin (“HbS”) less soluble and prone to polymerization upon deoxygenation (Hoffman, 2000). Erythrocytes (red blood cells, “RBC”) carrying polymerized HbS are thus less deformable and may obstruct microvessels. This vascular occlusion, producing tissue ischemia and infarction, represents a major cause of morbidity and mortality among sickle cell disease patients. Despite recent therapeutic advances with the use of hydroxyurea and butyrate (Charache, 1995; Atweh, 1999) many patients remain severely symptomatic and thus, may benefit from alternate therapeutic modalities.
Over the years, it has become clear that the clinical manifestations of sickle cell disease extend far beyond the homozygous globin mutation. Seminal findings were the discovery that sickle (“SS”) RBCs, unlike normal RBCs, could adhere to stimulated endothelium in vitro and that SS-RBCs' adhesion correlated with the clinical severity of sickle cell disease(Hoover, 1979; Hebbel, 1980 (a) and (b)). Subsequent studies have recognized the importance of plasma factors in SS-RBC adhesion to the endothelium (Wautier, 1983; Mohandas, 1984) and revealed that the deformable “low-density” cells were more adherent than the dense sickle-shaped cells (Mohandas, 1985; Barbarino, ). Other elegant studies by Kaul and coworkers subsequently showed using a rat mesocecum ex vivo perfusion model that SS-RBCs adhered exclusively in venules (mostly small post-capillary and collecting venules) and confirmed that adhesion was density-class dependent (light-density reticulocytes and young discocytes being most adherent; Kaul, 1989). Collectively, these observations lead to the current multistep model, shown in FIG. 1A, by which light-density SS-RBCs first adhere in post-capillary venules, after which secondary trapping of dense cells may produce vascular obstruction and local ischemia. These transient obstructions may induce HbS polymerization, which would increase RBC rigidity and exacerbate vascular occlusion.
Multiple adhesion molecules have been shown to participate in SS-RBC/endothelium interactions (FIG. 1B), Soluble adhesion molecules and matrix proteins were first recognized, and may function as a bridge between two cellular adhesion receptors or may recruit SS-RBCs directly to the vessel wall's matrix. These include fibrinogen and fibronectin (Wautier, 1983; Kasschau, 1996), von Willebrand factor (vWF; Wick, 1987; Kaul, 1993), laminin (Hillery, 1996; Lee, 1998) and thrombospondin (“TSP;Sugihara, 1992; Hillery, 1999). Several putative cellular counter-receptors have been suggested, although many are controversial or still poorly defined. For example, studies have suggested that TSP may interact with integrin associated protein (CD47; Gao, 1996) and sulfated glycolipids (Hillery, 1996), phosphatidylserine (Mandori, 2000) and CD36 (Sugihara, 1992) on the SS-RBC membrane. Other studies have suggested that CD36 is not involved in TSP-mediated sickle cell adhesion (Hillery, 1996; Joneckis, 1996). Membrane damage to SS-RBC with loss of phospholipid asymmetry (Frank, 1985) may expose phosphatidylserine as well as sulfated glycolipids which can interact with vWF and laminin (Roberts, 1986). Membrane damage to SS-RBC might also expose a portion of band 3 which may contribute to SS-RBC's adhesion with endothelial cells (Thevenin, 1997). Basal cell adhesion molecule/Lutheran protein (B-CAM-LU), the protein that carries the Lutheran blood group, was also shown to be a laminin receptor in SS-RBCs (Udani, 1998; Parsons, 2001). Finally, the integrin α4β1, one of the first sickle RBC adhesion receptor identified on sickle reticulocytes (Swerlick, 1993; Joneckis, 1993; Gee, 1995), can interact with vascular cell adhesion molecule-1 (“VCAM-1”) on activated endothelium. To date, few receptors for SS-RBCs have been identified on activated endothelium. In addition to VCAM-1 (Swerlick, 1993; Gee, 1995), α5β3 has been proposed to play an important role since functional inhibition of this receptor drastically reduced SS-RBC accumulation on platelet activating factor (“PAF”)-stimulated microvasculature in the ex vivo rat mesocecum (Kaul, 2000). Recent data also indicate that P-selectin may mediate SS-RBC adhesion to endothelial cells (Matsui, 2000). The foregoing studies of SS-RBC adhesion, however, suffer the shortcoming of having been performed in vitro or, in the case of Kaul, 2000, ex vivo; the mechanisms of vaso-occlusion had not, prior to the present invention, been explored in vivo.
Several mouse strains expressing HbS have been generated in the last decade. These transgenic strains have been used to study the pathophysiology of sickle cell disease in vivo, and may be divided into two broad categories: i) transgenic mice expressing both the endogenous murine and human globin genes, and (ii) transgenic mice expressing exclusively human globin genes (Nagel, 1998). So-called “SAD” mice represent one example of transgenic animal models for sickle cell disease in which the human β-globin transgene contains three natural mutations that enhance Hb sickling: HbS, HbS-Antilles and Hb D Punjab (hence the acronym “SAD”). RBCs from SAD mice carry approximately 19% human hemoglobin. Although associated with a significant perinatal mortality (when a SAD mouse is bred with a wild-type animal, the frequency of SAD offspring is about 30%, rather than the expected 50%), adult SAD transgenic mice are relatively healthy, suffering neither anemia nor reticulocytosis unless exposed to hypoxemic conditions (Trudel, 1991; Trudel, 1994). Transgenic “knock-outs” (hereinafter referred to as “sickle cell” or “SS” mice) were developed by sequential breeding of mice deficient in α and β globins with transgenic animals expressing both human a and βs globins; such SS mice are genetically identified as Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/−. These animals display a drastic phenotype characterized by severe anemia with high reticulocyte counts, splenomegaly and evidence of end-organ damage (Paszty, 1997; Ryan, 1997). Although the hematological and histological pictures in SS mice resemble that of patients, the phenotype in mice is more severe and their viability is reduced. When a male SS mouse is bred with a mouse heterozygous for β-globin expression (Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/+), less than 10% of the offspring exclusively express human globins, instead of the expected 50%. The reduced viability of SS mice has hampered the progression of in vivo studies and the development of useful models to evaluate the mechanisms of vaso-occlusion.
It had been noted, prior to the present invention, that sickle cell patients with leukocyte counts greater than 15,000/microliter have an increased risk of death (Platt, 1994), that lower neutrophil counts were associated with a lower crises rate in sickle cell patients treated with hydroxyurea (Churache, 1996) and that treatment with granulocyte colony stimulating factor (“G-CSF”, which increases leukocyte counts) induced a sickle cell crisis (Abboud, 1998). Schwartz, 1985, reported increased adherence of sickle RBCs to cultured peripheral blood monocytes in vitro, wherein irreversibly sickled RBCs and deoxygenated RBCs were most adherent and adhesion appeared to correlate with the exposure of phosphatidylserine to the outer membrane leaflet. Hofstra et al., 1996, reported that, in vitro, SS-RBCs can bind activated neutrophils in a static in vivo adhesion assay, an interaction which was more pronounced in the presence of autologous sickle cell plasma. Binding of SS-RBCs to activated neutrophils was partially inhibited by RGDS peptides and human IgG, suggesting than one or more integrin(s) and neutrophil Fc receptors may be involved. SS-RBC adhesion also induced an oxidative burst characterized by the production of free radicals by activated neutrophils (Id.) Further, it had been noted that anti-inflammatory agents such as methylprednisolone may be effective in decreasing the duration of sickle cell crisis episodes (Griffin, 1994). A recent study using a sickle cell mouse model indicated that the inflammatory response (number of adherent and emigrated leukocytes and oxidant production) resulting from hypoxia and reoxygenation was increased in sickle cell transgenic mice compared to control animals (Kaul, 2000).
Prior to the present invention, however, it had not been appreciated which of the many potential aspects of the inflammatory response was directly associated with vaso-occlusion.